Molecular Assay Used to Diagnose Buruli Ulcer.

The quick and inexpensive diagnostic test can identify Mycobacterium ulcerans, the bacterium responsible for Buruli Ulcer (BU), and could replace the current laboratory diagnosis based on microscopy, which has to be confirmed by polymerase chain reaction (PCR) and other tests in reference laboratories.

Scientists at the Noguchi Memorial Institute for Medical Research (Accra, Ghana) employed the methodology based on the loop mediated isothermal amplification (LAMP) technique. Four sets of Primers, targeting the mycolactone encoding plasmid genome sequence of M. ulcerans were designed. The BU-LAMP assay was developed and tested on five M. ulcerans strains from patients in Ghana and two reference isolates. The BU-LAMP assay protocol was optimized for maximum efficiency, using the Loopamp DNA amplification kit (Eiken Chemical; Tokyo, Japan).

The assay was tested on other closely related, mycolactone-producing mycobacterial strains; M. marinum, M. liflandii and M. pseudoshotsii, as well as experimentally infected laboratory animal and clinical samples. The results revealed a high specificity of the BU-LAMP assay for selectively detecting M. ulcerans. Compared to the conventional PCR, the new assay is cheaper, simpler, and ten times more sensitive. The test results can be obtained within one hour. The DNA concentration at the visual detection limit was estimated to be 48 pg/μL. However, by measuring the optical density (OD) value of BU-LAMP products at 350 and 450 nm, the scientists were able to detect positivity when using DNA concentrations as low as 0.5 fg/μL.

The authors concluded that the BU-LAMP method shows promise as a diagnostic tool at point of care facilities in BU endemic communities. Unlike the conventional PCR method that requires the use of a thermal cycler, purified DNA samples are not a requirement. DNA amplification occurs within one hour and the resulting product is a turbid solution, indicative of product amplification. Sample confirmation can therefore be done visually with the naked eye, and the intensity of the fluorescence observed is indicative of amount of DNA present. The study was first published online on January 12, 2012, in the journal BMC Infectious Diseases.

Related Links:
Noguchi Memorial Institute for Medical Research
Eiken Chemical